The high replication rate and large population size of some viruses requires both potent inhibitors and multiple inhibitors to achieve sustained inhibition of viral replication without the evolution of inhibitor resistance. Thus for viruses that cause chronic infections it is necessary to develop multiple drug targets. For HIV-1, seven targets have been exploited for drug discovery, at least to the point of clinical trials that have demonstrated some activity. Because of the extensive work that has been done with HIV-1, it is amenable to the exploration of new targets with the justification that new targets are needed to be able to treat viruses with multi-drug resistance to the existing inhibitors, and because these new targets could become broader paradigms for strategies to develop inhibitors of other viruses. We have identified what we believe is the most sensitive site in the HIV-1 replication cycle. Incomplete proteolytic cleavage at the MA/CA cleavage site in the HIV-1 Gag precursor leads to complete loss of virion infectivity even at very modest levels of uncleaved protein. This high sensitivity to under-cleavage is due to the fact that the CA domain must release from the membrane-bound MA domain and undergo a structural rearrangement involving the newly released CA N terminus in the context of the assembly of 2,000 CA molecules that form the mature virion core. A small amount of uncleaved MA/CA fusion protein is able to poison the structural integrity of the core resulting in an inability to carry out viral DNA synthesis in the subsequent round of infection. There is no other step in the viral life cycle where such a small amount of inhibition could ablate virion infectivity, making virion assembly the most sensitive step in the virus life cycle and cleavage of the MA/CA processing site the strongest validated transdominant mutation known. In this application we describe a fluorescence polarization assay that can monitor cleavage at the MA/CA processing site in the context of the folded protein substrate. We will validate this assay and use it in an initial screen of the LOPAC library, with secondary assays, to provide proof-of-principle for this new target. This work will result in the identification of a new class of HIV-1 inhibitors which will set the stage for the screening of larger libraries, and create a paradigm for identifying critical proteolytic processing events in the replication of other viruses to use as new targets. PUBLIC HEALTH RELEVANCE: A large number of drugs have been developed for HIV-1, but resistance remains a significant problem. Not only has that, but resistance to one drug frequently conferred some level of cross-resistance to other drugs directed at the same target. Thus there is a continuing need to develop new drug targets for HIV-1. This work will result in the identification of a new class of HIV-1 inhibitors which will set the stage for the screening of larger libraries, and create a paradigm for identifying critical proteolytic processing events in the replication of other viruses to use as new targets.